Materials and reagents

Biological materials

p-H3 (rabbit) (Ser10) antibody (Cell Signaling, catalog number: 9701S), store at -20 °C
Donkey anti-rabbit IgG (H+L), Alexa Fluor 555 (Life, catalog number: A31572)
100 ng/mL IGF-2 recombinant protein (R&D Systems, catalog number: 792-MG-050)
TNNT2^H2B-GFP/+mice (from Dr. Chen-Leng Cai, Background Strain: C57BL/6J)

Reagents

1× PBS pH 7.4 (Gibco, catalog number: 10010-023)
1 M HEPES (Gibco, catalog number: 15630-060), store at 4 °C
0.5 M EDTA pH 8.0 (Gibco, UltraPure, catalog number: 15575), store at 4 °C
1:250 Trypsin (Gibco, catalog number: 27250-018)
HBSS solution (Gibco, catalog number: 14175-095)
Horse serum (Gibco, catalog number: 16050-114), store at -80 °C
FBS (Gibco, catalog number: 10437-028), store at -80 °C
100× antibiotic-antimycotic (Gibco, catalog number: 15240-062)
1× DMEM [-] Ca²⁺(Gibco, catalog number: 21068-028), store at 4 °C
Laminin (Corning, catalog number: 354232), store at -80 °C
1× DPBS (Ca²⁺/Mg²⁺) (Corning, catalog number: 21-030-CV)
1× Opti-MEM (Gibco, catalog number: 31985-070), store at 4 °C
IC fixation buffer (Invitrogen, catalog number: 00822249), store at 4 °C
Triton X-100 (Fisher Biotech, catalog number: BP151-500)
Donkey serum (Fisher Brand, catalog number: 03-395-464)
0.1 μg/mL DAPI (1:1,000 working concentration) (Sigma, catalog number: D9542)
1× DMEM [+] Ca²⁺(Gibco, catalog number: 11965-092), store at 4 °C

Solutions

Trypsin solution (see Recipes)
Dissociation buffer (see Recipes)
Rinse media (see Recipes)
Culture media (see Recipes)

Recipes

Trypsin solution (in 1× HBSS)
Reagent Final concentration Quantity (25 mL)
HEPES (1 M) 10 mM 250 μL
EDTA (0.5 M) 0.54 mM 27 μL
Trypsin 0.25% 0.0625 g
HBSS solution (1×) n/a 25 mL
Dissociation buffer (in 1× HBSS) at 4 °C
Reagent Final concentration Quantity (25 mL)
Horse serum 10% 2.5 mL
FBS 5% 1.25 mL
HEPES (1 M)
HBSS Solution (1×)
n/a
n/a
250 μL
21.2 mL
Rinse media (in Ca²⁺free DMEM)
Reagent Final concentration Quantity (50 mL)
Horse serum 10% 5 mL
FBS 5% 2.5 mL
Antibiotic-antimycotic
Ca²⁺-free DMEM
1%
n/a
0.5 mL
42 mL
Culture media (in Opti-MEM)
Reagent Final concentration Quantity (20 mL)
Horse serum 10% 2 mL
FBS 5% 1 mL
Antibiotic-antimycotic
Opti-MEM
0.1%
n/a
20 μL
17 mL

Note: In isolating and plating the cardiomyocyte step, high quality and optimized concentration of FBS and horse serum are critical for cell viability and cell adherence.

Laboratory supplies

Surgical scissors (Roboz, catalog number: RS-5882)
Curved forceps (Stoelting, catalog number: 52102-54P)
7 mL transfer pipette (Globe Scientific, catalog number: 135038)
24-well culture plate (Denville, catalog number: T1024)
1.5 mL micro-centrifuge tube (Stellar Scientific, T3 Tube, catalog number: T17-300)
60 mm cell culture dish (Eppendorf, catalog number: 0030701111)
70 μm mesh cell strainer (Swish, catalog number: TC70-MT-3)
15 mL centrifuge tube (Denville, Thomas Scientific, catalog number: 1158R11)

Equipment

M205 FCA microscope (Leica)
Hybridization oven/shaker (Amersham Biosciences)
Centrifuge 5425 R (Eppendorf)
TSX60086A -80 °C freezer (Thermo Scientific)
Isotemp CO₂incubator (Fisher brand)
Centrifuge 5702 (Eppendorf)
Handheld automated cell counter (Millipore, Scepter 3.0, catalog number: PHCC30000)
60 μm sensors (Millipore, Scepter 3.0, PHCC360050)
-20 °C freezer (Fisher Scientific, Isotemp)
4 °C fridge (Insignia)
BZ-X800 microscope (Keyence)
Standard orbital shaker, Model 5000 (VWR, catalog number: 89032-100)

Software and datasets

ImageJ (https://imagej.nih.gov/ij/download.html)

Procedure

Cardiomyocyte isolation
1. Collect GFP-positive mutant hearts from E13.5–E14.5 embryos (see General note 1).
  1. Make the solutions for the experiment and prepare a sterile workspace. Ensure that the PBS is refrigerated ahead of time and kept on ice throughout the duration of the procedure.
  2. Anesthetize dam at E13.5–E14.5 in accordance with IACUC guidelines.
  3. Use a scalpel to make an abdominal incision and extract the embryos with curved forceps. Separate the embryo from the placenta and yolk sac with surgical scissors. Gently remove each heart (see General note 2) using curved forceps and use a transfer pipette to move it into a 24-well plate containing cold PBS. Each heart should be placed in an individually labeled well.
  4. Check the hearts for fluorescence under a fluorescence-emitting microscope (Figure 1) and label the wells accordingly.
    
    Figure 1. Embryonic day (E) 13.5 hearts from the same litter (TNNT2^H2B-GFP/+) imaged in brightfield and GFP fluorescence at 10× magnification. Compared to wild type (above), the TNNT2^H2B-GFP/+mouse (below) has a positive indicator for the GFP-tagged TNNT2 gene. Scale bar, 1 mm.
2. Isolate cells (see General note 3).
  1. Using a transfer pipette, move each GFP-positive sample from the 24-well plate to a 1.5 mL microcentrifuge tube. All GFP-positive samples from the litter will be pooled into the same tube and digested together. Then, pipette out excess PBS with a micropipette. Add 500 μL of trypsin solution (see Recipes) to the tube and gently pipette the solution up and down with the micropipette to mix.
    Note: Each heart can be dissociated individually per embryonic age and experimental needs (see General note 4).
  2. Use tape to secure the microcentrifuge tube to the platform of a hybridization shaker. Ensure that it is positioned such that the solution can move throughout the tube and mix properly. Shake at 70 strokes/min at 37 °C for 20 min.
  3. Remove the tube from the shaker and gently pipette the liquid up and down with a 1 mL pipette 10–15 times until there are no observable tissue pieces.
  4. Transfer the liquid from the 1.5 mL microcentrifuge tube to a 15 mL centrifuge tube using the same micropipette. Add 5 mL of cold (4 °C) dissociation buffer (see Recipes) to the 15 mL centrifuge tube. Pipette up and down with a 7 mL transfer pipette until the cells are disaggregated in the solution.
  5. Centrifuge at 400×gfor 5 min at 4 °C.
  6. In a sterile vent-hood with sanitized surfaces to avoid contamination, vacuum-pipette and discard the resulting supernatant from the tube.
  7. Add 1 mL of rinse media in Ca²⁺free DMEM (see Recipes) with a micropipette.
  8. Centrifuge at 400×gfor 5 min at 4 °C.
  9. Vacuum-pipette and discard the resulting supernatant from the tube. Add 1 mL of culture media in Ca²⁺free DMEM (see Recipes) with a micropipette.
  10. Use a new micropipette to disaggregate the pellet and centrifuge again (400×gfor 5 min at 4 °C). The resulting supernatant will be used for plating and should not be discarded.
    Protocol continues in step B2.
Plating cells in culture media (see General note 3)
1. Laminin coating
  Note: This step should be completed the day before plating.
  1. To avoid damaging the laminin via excessive freezing and thawing, pipette out laminin (final concentration: 10 μg/mL) with a micropipette and transfer to a 1.5 mL microcentrifuge tube. Return the remaining laminin to a -80 °C freezer.
  2. Dilute the laminin with 1× DPBS (Ca²⁺/Mg²⁺) (see General note 5) and add the solution to a 24-well culture plate (see General note 6). Ensure the entire surface is covered by the laminin coating solution.
  3. Seal the plate with parafilm to prevent evaporation and contamination.
  4. Incubate at 2–8 °C overnight. If a more rapid coating is required, incubate at 37 °C for 2 h.
2. Plating the cardiomyocytes
  1. Using a 5 mL serological pipette attachment for an assisted pipette controller, add 4 mL of culture media to an uncoated 60 mm cell culture dish.
  2. Through a 70 μm mesh cell strainer, add 1 mL of the supernatant from the microcentrifuge tube to the cell culture dish.
  3. Check that the cells are present in the dish under fluorescence microscope (Figure 2).
    
    Figure 2. Visualization of cells collected from embryonic day (E) 13.5 hearts at 20× magnification plated in a 48-well cell culture plate with culture media. Brightfield imaging shows all the heart cells present in the dish, whereas GFP fluorescence identifies cardiomyocytes via the H2B-GFP tag. The combined brightfield/GFP image illustrates the ratio of non-cardiomyocyte to cardiomyocyte cells in the embryonic heart, necessitating the subsequent laminin-plating step for isolating cardiomyocytes. Scale bar, 100 μm.
  4. Pre-plate the dish for 1 h in a cell culture incubator at 37 °C in 5% CO₂to allow nonmyocytes (fibroblasts and endothelial cells) to adhere to the dish.
  5. Centrifuge the whole dish in the Eppendorf 5702 centrifuge with bucket rotors at 400×g for 5 min.
  6. Collect the suspended cells carefully to avoid adherent cells, as they are primarily non-cardiomyocytes (e.g., fibroblasts and endothelial cells).
  7. Load cells into a 60 μm sensor and run through the Scepter 3.0 handheld automated cell counter. The output will determine the size of wells needed for the laminin plating.
  8. Use a vacuum pipette to remove excess laminin solution from the premade 24-well plate.
  9. Transfer the cells to the premade laminin-coated plate (10 μg/cm²).
  10. Incubate at 37 °C in 5% CO₂.
  11. After 24 h, cardiomyocytes can be seen beating under a microscope (Video 1) (see General note 7). Remove the solution and replace it with serum-free media (DMEM) for one hour to starve the cells.
    
    Video 1. Embryonic day (E) 13.5TNNT2^H2B-GFP/+cardiomyocytes beating after 24 h in culture media
  12. Wash the cells with PBS.
  13. Treat the cells with 100 ng/mL recombinant IGF-2 protein growth factors.
Immunocytochemistry and cell counting (see General note 8)
1. Wash the cells with ice-cold PBS.
2. Fixate cells using 200 μL per well of IC fixation buffer for 10 min at room temperature.
3. Wash the cells three times with room temperature PBS for five minutes.
4. Permeabilize the sample by adding 50 µL (or enough to cover the cells) of PBS-T (PBS containing 0.1% Triton X-100) per well for 10 min at room temperature.
5. Wash the cells three times with room temperature PBS for five minutes.
6. Incubate cells with 50 µL (or enough to cover the cells) of 5% donkey serum in PBS per well for 1 h to block non-specific antibody binding.
7. Incubate cells with 50 µL (or enough to cover the cells) of 1:250 diluted primary antibody [p-H3 (rabbit) - donkey anti-rabbit] in 1% donkey serum in PBS per well overnight at 4 °C.
8. Decant the solution and wash the cells three times in PBS for five minutes.
9. Incubate cells with 50 µL (or enough to cover the cells) of 1:500 diluted secondary antibody [donkey anti-rabbit IgG (H+L), Alexa Fluor 555] in 1% donkey serum in PBS per well for 1 h at room temperature in the dark.
10. Decant the secondary antibody solution and wash the cells three times in PBS for five minutes in the dark.
11. Incubate cells in 50 µL (or enough to cover the cells) of 0.1 μg/mL 1:1,000 DAPI working concentration per well for 1 min.
12. Remove DAPI and add 200 µL of PBS to each well.
13. Image cell plate at 20× with Keyence microscope (Figure 3).
  
  Figure 3. Fluorescence imaging at 20× magnification of p-H3 immunocytochemistry-stained embryonic day (E) 13.5 cardiomyocytes in serum-free media (above) and IGF-2 recombinant protein (below). When exposed to GFP fluorescence, H2B-GFP tagged cardiomyocytes are visible (column 1). Proliferating cells are identified with the p-H3 stain (column 2). The combined GFP/p-H3 image (columns 3 and 4) allows for quantification of GFP+p-H3+ vs. only GFP+ cardiomyocytes in serum free and IGF-2-treated E13.5 heart cells. Scale bar, 275 μm. Percentage of p-H3+ cardiomyocytes and total number of cardiomyocytes were quantitated. IGF2, n = 4 in each group. P-values were determined by the Mann-Whitney U test.
14. Both the total number of cells (Green) and pH3+ cells (Red) are counted by ImageJ image processing software. For total cardiomyocyte number, we counted GFP-positive cardiomyocyte cells in ImageJ. For percentage of p-H3 within total cardiomyocytes, we counted each of p-H3-positive cells and GFP-positive cells.

General notes and troubleshooting

General notes

E13.5 is the ideal collection day for cardiomyocytes. If the hearts are collected prior to E12.5, there may not be enough cells, whereas if the hearts are collected after E14.5 there will be too many fibroblasts present in the samples.
As fluorescence cannot be observed in the whole embryo, hearts must first be removed to check fluorescence. For the purposes of this study, the whole heart was collected, but it is possible at this stage to separate the atria or ventricles if necessary. Additionally, this protocol can be used in wildtype (WT) hearts by staining cardiomyocyte markers such as cardiac Troponin T. GFP+ cell sorting by FACS can be performed before plating the cells.
All cell culture steps require sanitized gloved hands and surfaces in a sterile fume hood to prevent contamination.
As E13.5 hearts are approximately 1–2 mm in size, 500 μL of trypsin solution is enough to digest as many hearts as are collected from one litter.
DPBS with Ca²⁺and Mg²⁺should be used to dilute the laminin, since divalent cations are important for protein structure and function.
Based on the number of cells present in the pre-plated cell culture dish, the 24-well plate can be replaced with a 48-well plate (for 1 × 10⁴cells) or a 96-well plate (for 4 × 10⁴cells).
Twenty-four hours after plating the isolated cells, cardiomyocytes visibly beat and will reach confluency 48 h later. In this protocol, we treated the recombinant protein under serum-free conditions and did not passage the cells over seven days. However, in normal serum conditions, we can observe the beating cardiomyocytes 14 days after isolating the cells from the heart.
For each wash step, add approximately 200 μL per well or enough to cover the cells and remove after 5 min with a vacuum pipette; then, proceed to the next step with haste to prevent the cells from drying out. Steps for the immunocytochemistry procedure are performed on a standard orbital shaker.

Acknowledgments

We gratefully acknowledge Dr. Chen-Leng Cai (Indiana University, Indianapolis, Indiana, USA) for providingTNNT2^H2B-GFP/+mice. This work was partially supported by American Heart Association-Career Development Award 23CDA1046244 (J.J.), the National Heart, Lung, and Blood Institute R01 grant HL153406 (D.L.) and American Heart Association-Transformational Project Award 20TPA35490132 (D.L.). Graphical overview created with BioRender.com.

Competing interests

There are no conflicts of interest or competing interests.

Ethical considerations

Tnnt2^H2B-GFP/+mice were previously described. Animal protocol was approved by the Nationwide Children’s Institutional Animal Care and Use Committee (IACUC).

References

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Rodgers, L. S., Schnurr, D. C., Broka, D. and Camenisch, T. D. (2009). An improved protocol for the isolation and cultivation of embryonic mouse myocytes. Cytotechnology 59(2): 93–102.
Yan, J., Zhang, L., Sultana, N., Oh, J. G., Wu, B., Hajjar, R. J., Zhou, B. and Cai, C. L. (2016). A series of robust genetic indicators for definitive identification of cardiomyocytes. J. Mol. Cell. Cardiol. 97: 278–285.
Zaidi, S. and Brueckner, M. (2017). Genetics and Genomics of Congenital Heart Disease. Circ. Res. 120(6): 923–940.